Presentation on theme: "S ELECTED A ROMATIC A MINES BY G AS C HROMATOGRAPHY M ASS S PECTROMETRY : C HALLENGES OF M AINSTREAM C IGARETTE S MOKE Alexandra Martin October 3, 2013."— Presentation transcript:
S ELECTED A ROMATIC A MINES BY G AS C HROMATOGRAPHY M ASS S PECTROMETRY : C HALLENGES OF M AINSTREAM C IGARETTE S MOKE Alexandra Martin October 3, 2013
What are PAAs and why are they important? Smoke Collection 101 Brief history of PAA analysis New challenges - FDA HPHC list SPE clean-up GC-MS parameters – EI vs. NCI Validation results LOD/LOQ Accuracy Precision O UTLINE
W HAT A RE PAA S AND W HY A RE T HEY I MPORTANT ?
B ACKGROUND Tobacco smoke contains more than 4,000 chemical compounds. Characterization of this matrix has been the focus of scientists for many years. One of the goals of this work has been to identify the compounds primarily responsible for serious health effects. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans - Classification
PAA C LASSIFICATIONS AND R EPORTING R EQUIREMENTS Compound IARC Class Health Canada Brazil ANVISA US FDA ortho -toluidine1 X 2,6-dimethylaniline2B X ortho -anisidine2B X 1-aminonaphthalene3 XXX 2-aminonaphthalene1 XXX 3-aminobiphenylna XX 4-aminobiphenyl1 XXX
T HE O RIGINAL F OUR PAA S 1-aminonaphthalene2-aminonaphthalene 3-aminobiphenyl 4-aminobiphenyl
A DDITIONS FOR FDA HPHC L IST 2,6-dimethylaniline o -anisidine o -toluidine
A MERICAN H EALTH F OUNDATION M ETHOD “On the Analysis of Aromatic Amines in Tobacco Smoke”. C. Patrianakos, K.D. Brunnemann and D. Hoffmann, American Health Foundation, Valhalla New York 10595. Presented at the 31 st TCRC, Greensboro, NC, October 1977. C. Patrianakos and D. Hoffmann, “Chemical Studies on Tobacco Smoke LXIV. On the Analysis of Aromatic Amines in Cigarette Smoke”, J. Anal. Toxicol., 3, 150-154 (1979).
AHF M ETHOD (1977) C ONTINUED … Florisil – 10 g Fraction (75 mL), Evaporate Florisil – 30 g Fraction 1 (500 mL) and Fraction 2 (400 mL) GC-MS or GC-ECD Analysis Wash, Dry, Evaporate Evaporate each to 2 mL
H EALTH C ANADA M ETHOD Health Canada Official Test Method T-102, 1999 Based upon Pieraccini et al 1992 Modifications CFP for trapping, not impinger Smaller volume solvent used Fewer evaporation steps Much better LOQs G.Pieraccini, F. Luceri and G. Moneti, “New Gas-chromatographic/Mass Spectrometric Method for the Quantitative Analysis of Primary Aromatic Amines in Main- and Side-stream Cigarette Smoke. I”, Rapid Communications in Mass Spectrometry, 6, 406-409 (1992).
G OALS OF N EW M ETHOD One method to determine all 7 PAAs. No evaporation steps. Evaluate SPE clean-up to reduce solvent use and improve reproducibility and efficiency of method. Retain the advantages of NCI (lower detection limits, improved selectivity).
N EW S AMPLE P REPARATION S CHEME (A RISTA M ETHOD ) Smoke Collection (CFP) Extraction of CFP w/ 5% HCl [Add ISTDs] SPE Clean-Up and Solvent Exchange (DCM) Derivatize (PFPA) GC-MS Analysis (NCI)
Selectivity Can selectively retain/release compounds of interest. Requires small volumes of solvent New method uses 8-10 mL vs. 60-70 mL with liquid:liquid. Efficiency Can process many more samples in a batch with manifolds of up to 24 ports. Effectively isolates and concentrates analytes of interest! SPE A DVANTAGES
W HY IS C LEAN - UP I MPORTANT ? Response Interferences can swamp the system and reduce sensitivity. Sample Throughput Dirty samples can destroy the chromatography and suppress the detector response and can increase down time due to system maintenance. Quantitation With GC/MS by NCI PAAs have simple spectra - quantitation is based on one significant daughter ion. Clean-up and separation are critical!
D ETECTION - EI Electron Ionization M + e - → M + + 2e - M + produces a characteristic fragmentation pattern or a “mass spectrum” specific to each compound.
D ETECTION – NCI Chemical Ionization (methane) CH 4 + e - → CH 4 + + 2e - The electrons have much lower energies. Most compounds cannot form stable negative ions but react with the CH 4 + (PCI). If a compound can capture and hold an extra electron and retain a negative charge, it can be observed in NCI mode. MF + e - → MF - → [M-H] - + HF
C HROMATOGRAM OF L OW S TANDARD (EI VS. NCI) EI
C HROMATOGRAM OF L OW S TANDARD (EI VS. NCI) EI NCI
C HROMATOGRAMS OF A C IGAR E XTRACT (EI VS. NCI) 1-aminonaphthalene 2-aminonaphthalene 3- aminobiphenyl 4-aminobiphenyl o -toluidine o - anisidine 2,6-DMA o -toluidine 2,6-DMA o - anisidine 1-aminonaphthalene 2-aminonaphthalene 3- aminobiphenyl 4-aminobiphenyl EI NCI
M ETHOD C OMPARISON (W E ’ VE C OME A L ONG W AY, B ABY ) AHF (1977) HC T- 102 (1999) Arista (2012) No. of Cigarettes210105 Volume Organic Solvent (mL) > 3000~ 3006 – 8 No. of SPE Steps212 No. of Evaporation Steps42None Final Volume (mL)215 ~ 4-AMB LOQ (ng/mL)500.50.01
LCMSMS J. Schubert, O. Kappenstein, A. Luch and T. G. Schulz, “Analysis of primary aromatic amines in mainstream waterpipe smoke using liquid chromatography-electrospray ionization tandem mass spectrometry”, J. Chrom. A, 1218, 5628-5637 (2011).
S UMMARY Achieving lower and lower detection limits in complicated matrix requires both improving clean-up (SPE) and selectivity of the instrument (NCI). The method presented here demonstrates excellent detection limits, selectivity, accuracy and precision. It is robust, efficient and fit-for-purpose, accommodating the regulatory requirements of Health Canada, ANVISA and the US FDA.